The Myc family of oncoproteins (c-, N-, and L-Myc) have been implicated in a wide range of cellular behavior including cell cycle entry and maintenance, differentiation, and apoptosis. In addition rearrangements at myc gene loci have been associated with many types of neoplasia in numerous species, including humans. Despite a vast amount of information on the properties and biological effects of Myc we lack a direct molecular link between these cellular processes and the Myc oncoprotein itself. Recently Max, a specific Myc dimerization partner, has been identified which forms a sequence-specific DNA binding complex with Myc. The Myc:Max complex appears to function as a transcriptional activator and evidence suggests that the relative abundances of constitutively expressed Max protein and highly regulated Myc protein may serve to determine the activity of this transcriptional 'switch'. In this application we propose to extend our studies on Myc and Max and to examine in detail the biochemical and biological properties of a new Max binding protein, Mad. Mad competes with Myc for binding to Max and appears to act as a repressor which can antagonize Myc's transcriptional activation function. This suggests that Myc, Max, and Mad may form a small network of interacting proteins. To explore the dynamics of this network we plan to characterize the properties and interactions of Mad in vivo and determine whether it may negatively influence Myc's transforming activity and act as a tumor suppressor gene. We will also probe the effects of both Max and Mad expression on the G0/G1 transition, cell proliferation, differentiation, and apoptosis. Biological roles for Mad in development will be studied by means of targeted gene disruptions in mice. Another series of experiments will examine the transcriptional properties of myc, Mad, and Max by analyzing, through site specific mutagenesis, the domains of these proteins that contribute to transcription activation and repression. The effects of alternative binding sites and promoter structure will also be analyzed. An in vitro transcription system will be developed to study the biochemical properties of these proteins in greater detail and the effects of other transcription factors on their activities. Finally we will attempt to extend our knowledge of the network of protein interactions involved in Myc function by using interaction cloning strategies to identify new binding partners for Myc, Max and Mad. The information gained from the studies proposed here should enable us to understand both how Myc functions at the molecular level and the manner in which this function is regulated.